Intradermal delivery of active agents by needle-free injection and electroporation

ABSTRACT

Methods are proved for introducing a biologically active agent into cells of a subject by introducing the agent in a form suitable for electrotransport into a region of tissue of the subject using one or more needle-free injectors, and applying a pulsed electric field to the region of tissue, thereby causing electroporation of the region of tissue. The combination of needle-free injection and electroporation is sufficient to introduce the agent into cells in skin, muscle or mucosa. For example, the region of tissue can be contacted with two oppositely charged injectors, one acting as the donor electrode and one acting as the counter electrode, or a single injector and one or more electrodes can be used. In addition, needle-free injection may be used in combination with suitable non-invasive electrode configurations. The active agents delivered into cells using the invention method can be small molecules, polynucleotides, polypeptides, and the like.

RELATED APPLICATION

This application is a Continuation-In-Part application of U.S. Ser. No.09/567,404, filed May 8, 2000, now U.S. Pat. No. 6,520,950 which reliesfor priority under 35 U.S.C. § 119(e), upon U.S. Provisional ApplicationSer. No. 60/133,265, filed May 10, 1999, each of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

This invention generally relates to methods for delivery of an activeagent to a subject and more specifically to the use of electroporationand needle-free delivery of an active agent to a subject.

BACKGROUND

A cell has a natural resistance to the passage of molecules through itsmembranes into the cell cytoplasm. Scientists in the 1970's firstdiscovered “electroporation,” the use of electrical fields to createpores in cells without causing permanent damage to the cells. Thisdiscovery made possible the insertion of large molecules directly intocell cytoplasm. Electroporation was further developed to aid in theinsertion of various molecules into cell cytoplasm by temporarilycreating pores in the cells through which the molecules pass into thecell.

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the agent to beintroduced therein and placed between electrodes, such as parallelplates. Then, the electrodes are used to apply an electrical field tothe mixture containing the cells and the agent to be introduced therein.

With in vivo applications of electroporation, electrodes are provided invarious configurations such as, for example, a caliper that grips theepidermis overlying a region of cells to be treated. Alternatively,needle-shaped electrodes may be inserted into the patient, to accessmore deeply located cells. In either case, before, simultaneously, orafter the agent is injected into the treatment region, the electrodesare used to apply an electrical field to the region. See, for example,U.S. Pat. No. 5,019,034, issued May 28, 1991 and U.S. Pat. No.5,702,359, issued Dec. 30, 1997.

Electroporation (both in vitro and in vivo) functions by causing cellmembranes to which a brief high voltage pulse is administered totemporarily become porous, whereupon molecules can enter the cells. Insome electroporation applications, the electric field comprises a singlesquare wave pulse on the order of 1000 V/cm, of about 100 microsecondsduration. Such a pulse may be generated, for example, in knownapplications of the ElectroSquarePorator T820, made by the BTX Divisionof Genetronics, Inc.

Electroporation has been recently suggested as an alternate approach tothe treatment of certain diseases such as cancer by introducing achemotherapeutic drug directly into the cell. For example, in thetreatment of certain types of cancer with chemotherapy it is necessaryto use a high enough dose of a drug to kill the cancer cells withoutkilling an unacceptably high number of normal cells. If the chemotherapydrug could be inserted directly inside the cancer cells, this objectivecould be achieved. However, some of the best anti-cancer drugs, forexample, bleomycin, cannot penetrate the membranes of certain cancercells effectively under normal circumstances. To overcome thisdifficulty, electroporation has been used to cause bleomycin topenetrate the membranes of cancer cells.

Electroporation-assisted chemotherapy typically is carried out byinjecting an anticancer drug directly into the tumor and applying anelectric field to the tissue between a pair of electrodes. The fieldstrength must be adjusted reasonably accurately so that electroporationof tumor cells occurs without damage, or at least with minimal damage,to any normal or healthy cells. Typically, this method is employed withtumors located on the exterior of the patient's body by applyingelectrodes to the body surface on opposite sides of the tumor, thuscreating an electric field between the electrodes. When the field isuniform, the distance between the electrodes can then be measured and asuitable voltage, derived according to the formula E=V/d (whereinE=electric field strength in V/cm; V=voltage in Volts; and d=distance incm), can then be applied to the electrodes. However, when the tumors tobe treated are large, irregular in shape, or located within the bodyinterior, it is more difficult to properly locate electrodes and measurethe distance between them so as to accurately calculate the voltage thatis to be applied. In such cases, needle array electrodes as, forexample, described in U.S. Pat. No. 5,993,434 (Dev and Hofmann) haveproven to be advantageous.

Using these and related techniques (for example, the molecule can bedelivered encapsulated in a liposome), electroporation has been used todeliver molecules into many different types of cells. For example,electroporation has been used to deliver biologically active agents tovarious human and mammalian cells, such as egg cells (i.e., oocytes),sperm, platelets, muscle, liver, skin, and red blood cells. In addition,electroporation has been used to deliver molecules to plant protoplasts,plant pollen, bacteria, fungi, and yeast. A variety of differentbiologically active molecules and agents have been delivered to cellsusing this technique, including DNA, RNA and various chemical agents.

Vaccination is the most cost-effective way to prevent disease. However,there are still many diseases for which no vaccine exists or for whichthe currently available vaccines are inadequate. DNA immunizations,which entail the administration of DNA encoding an antigen, may offersolutions in at least some of these cases. Moreover, DNA vaccines offerthe use of host cells as bioreactors for the production of proteins invivo (Tang, D. C., et al., (1992) Nature 356:152–4). By doing so, DNAvaccines mimic a viral infection, improve antigen presentation to theimmune system relative to standard protein vaccines, and work moreeffectively as a result (Ulmer, J. B., et al., (1993) Science259:1745–9). Moreover, DNA vaccines offer these potential benefitswithout many of the safety and stability concerns associated with theadministration of infectious agents.

DNA immunization has been effective in several small animal models(Donnelly, J. J., et al., (1997) Annu. Rev. Immunol. 15:617–48).However, demonstrating its effectiveness has been much more challengingin larger animals and humans. Numerous studies have shown that thegreatest power of DNA vaccines may be their ability to prime the immunesystem for responses to other vaccines (Richmond, J. F., et al., (1998)J. Virol. 72:9092–100; Robinson, H. L., et al., (1999) Nat. Med.5:526–34).

The first hypodermic syringe was developed by a French surgeon,Charles-Gabriel Pravaz, in 1853 to take advantage of the highlypermeable interstitial tissue below the skin surface to transportpharmaceuticals to active sites. Although there have been developmentsin hypodermic syringes since then, the technology has remainedessentially unchanged for the past 150 years. Needle-free injection wasdeveloped when workers on hydraulic equipment noticed that high-pressuresquirts of hydraulic oil would pierce the skin. The first description ofneedle-free injection was in Marshall Lockhart's 1936 patent for “jetinjection.” Then, in the early 1940's Higson and others developedhigh-pressure “guns” using a very fine jet of liquid medicament topierce the skin and deposit it into the tissue underneath. In World WarII, needle-free guns were used extensively to inoculate troops en masseagainst infectious disease. Later, needle-free guns were applied moregenerally in large-scale vaccination programs.

However, these early needle-free injectors were used on multiplepatients and fears about the transmission of hepatitis B and HIVinfection by reuse of the injectors led to a sharp decline in their use.Until recently, the main application of such devices was veterinary,with a few being used by diabetics for self-treatment.

In the past 50 years, over 300 patents have been filed in theneedle-free delivery area. Although various improved products have cometo the market, none has gained wide use and remnants of the olderdevices remain to this day. These devices tend to be expensive topurchase and difficult to use, requiring the user to perform a series ofcomplicated steps to set up the device for use. For example, some ofthese systems require the user to fit a needle to the delivery devicetemporarily in order to draw liquid containing the desired active agentinto the device from a vial. Therefore, even the more modern needle-freedelivery systems do not address the needs of the market for an easy touse, low cost, and simple system. Consequently, needle-free delivery hasnot come into widespread use.

Despite this apparent failure of needle-free delivery, thepharmacokinetics and pharmacodynamics of needle-free delivery are welldocumented. Accelerating a jet of liquid to high speed provides powerfor the liquid to penetrate the stratum corneum as well as individualcell membranes. Thus, there is a need in the art for new and bettermethods for transporting molecules, such as biologically active agents,across the stratum corneum and/or cell membranes in treatment of avariety of conditions and diseases.

SUMMARY OF THE INVENTION

The present invention overcomes such problems in the art by providingmethods for introducing biologically active agents into cells withoutuse of a hypodermic needle. In one embodiment according to the presentinvention, a biologically active agent is introduced in a form suitablefor direct or indirect electrotransport into a region of tissue of thesubject using one or more needle-free injectors, and an electric fieldis applied to the region of tissue, thereby causing electroporation ofthe region of tissue prior to, simultaneously with, and/or subsequentlyto introducing the agent. Direct electrotransport refers to thetransport of molecules subjected to an electrical or magnetic force,indirect electrotransport refers to the transport of moleculesfacilitated by electric forces which act primarily on transportbarriers, e.g., cell membranes, which become more permeable as a resultof electric forces. The combination of needle-free injection andelectroporation is sufficient to introduce the active agent into thecell and allows for delivery of pharmaceutical compounds, nucleic acidconstructs, or other agents into cells contained within the tissueregion so treated.

In another embodiment according to the present invention, a biologicallyactive agent is introduced into cells in a region of tissue of a subjectby contacting the region of tissue or adjacent tissue with two or morespaced apart needle-free injectors while injecting a biologically activeagent into the tissue, and applying an electrical field to the tissuevia the two or more injectors prior to, simultaneously with, and/orsubsequently to injection of the agent so as to electroporate the regionof tissue, whereby the combination of needle-free injection andelectroporation is sufficient to introduce the agent into the cell.

In yet another embodiment according to the present invention, abiologically active agent is introduced into cells in a region of tissueof a subject by contacting the region of tissue with at least oneneedle-free injector while injecting an agent suitable for direct orindirect electrotransport into the region of tissue, and applying anelectrical field across the region of tissue using the at least oneinjector prior to, simultaneously with, and/or subsequently to injectionof the agent, whereby the combination of needle-free injection andelectroporation is sufficient to introduce the agent into the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B show diagrams illustrating the invention method whereinelectrically conducting needle-free injectors are used as the electrodesfor delivering an electrical impulse to a region of tissue. In FIG. 1A,two needle-free injectors are disposed in spaced apart relation to oneanother and in contact with the surface of a region of tissue of thesubject. The oppositely charged injectors act as electrodes forconducting electroporation, being connected with an electrical source,such as a pulse generator, such that an electrical current is deliveredthrough the region of tissue by completing the circuit between the twoelectrically conducting injector tips. One injector is the active ordonor electrode and the second, oppositely charged, injector is thecounter or return electrode. In FIG. 1B, one needle-free injectorcontacts the surface of a region of tissue while providing an electricalcurrent in conjunction with two oppositely charged electrodes. Theinjector acts as the active or donor electrode and the two ringelectrodes act as counter or return electrodes.

FIG. 2A is a schematic drawing showing a needle-free injector that isnot in contact with the skin injecting a liquid into tissue through anopening in an array electrode containing multiple positive and negativeelectrodes.

FIG. 2B is a schematic drawing showing a needle-free injector with arrayelectrode attached to the nozzle area and an opening in the arrayelectrode allowing the liquid jet to go through the electrode into theskin.

FIG. 2C is a schematic drawing showing a needle-free injector with anarray electrode attached to the nozzle area. The array electrode hasmultiple openings to allow multiple liquid jets to pass through thearray electrode into the skin.

FIG. 3 is a graph showing electroporation used in conjunction withintradermal injection of DNA vaccine improves gene expression levels.Intradermal expression levels are shown for DNA vaccine injected usingneedle-free BioJect (b.j.) and needle (i.d.n.) injections, without orwith electroporation, at the voltages indicated. Error bars representstandard error of the mean (SEM).

FIG. 4 is a graph showing immune responses to Hepatitis B after variouscombinations of DNA vaccine followed by protein booster immunizations.HBsAg antibody titers at 10 weeks were determined using the AUSAB EIAand titers shown are the geometric mean of 5 animals for all the groupsexcept for the i.m. subunit group, which had 4 animals. Error bars areSEM.

FIGS. 5A–5D are graphs showing antibody isotype responses elicited byHepatitis B immunizations according to the invention methods. Datarepresent Hepatitis B specific IgG1 and IgG2 titers at 8 and 10 weeksfor individual animals. The bar represents the geometric mean. Group1=needle-free injection of DNA, Group 2=needle-free injection ofDNA+electroporation (EP), Group 3=intradermal injection of DNA, Group4=intradermal injection of DNA+EP, Group 5=needle-free administration ofthe subunit vaccine control, and Group 6=intramuscular injection of thesubunit vaccine control.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, there are provided methods forintroducing a biologically active agent into cells in a region of tissueof a subject by injecting the agent in a form suitable for direct orindirect electrotransport into a region of tissue of the subject usingone or more needle-free injectors, and applying an electric field to theregion of tissue, thereby causing electroporation of the region oftissue prior to, simultaneously with, and/or subsequently to injectionof the agent. The combination of needle-free injection andelectroporation is sufficient to introduce the agent into the cell.

A “needle-free injector,” as the term is used herein, refers to a devicethat injects an agent into tissue without the use of a needle, forexample as a small stream or jet, with such force (usually provided byexpansion of a compressed gas, such as carbon dioxide through amicro-orifice within a fraction of a second) that the agent pierces thesurface of the tissue and enters underlying tissue and/or muscle. In oneembodiment, the injector creates a very high-speed jet of liquid thatpainlessly pierces the tissue. Such needle-free injectors arecommercially available and can be used by those having ordinary skill inthe art to introduce agents (i.e. by injection) into tissues of asubject. Examples of needle-free injectors that can be utilized inpractice of the invention methods include those described in U.S. Pat.Nos. 3,805,783; 4,447,223; 5,505,697; and 4,342,310.

As used herein, the term “introduce,” “inject” or “injecting,” andgrammatical equivalents thereof, as applied to the action of aneedle-free injector means that the agent is forced through at least thesurface of the tissue (e.g., the mucosa or epidermis, stratum corneum,or dermis of skin) and, preferably, delivered into underlying tissueand/or musculature using a needle-free injector as described herein.

A desired agent in a form suitable for direct or indirectelectrotransport is introduced (e.g., injected) using a needle-freeinjector into the tissue to be treated, usually by contacting the tissuesurface with the injector so as to actuate delivery of a jet of theagent, with sufficient force to cause penetration of the agent into thetissue. For example, if the tissue to be treated is mucosa, skin ormuscle, the agent is projected towards the mucosal or skin surface withsufficient force to cause the agent to penetrate through the stratumcorneum and into dermal layers, or into underlying tissue and muscle,respectively.

Needle-free injectors are well suited to deliver active agents to alltypes of tissues, particularly to skin and mucosa. In some embodiments,a needle-free injector may be used to propel a liquid that contains DNAmolecules or a drug toward the surface and into the subject's skin ormucosa. Representative examples of the various types of tissues that canbe treated using the invention methods include pancreas, larynx,nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney,muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue,ovary, blood vessels, or any combination thereof.

In addition to their function in introducing the active agent, two ormore needle-free injectors can also be used to apply an electric fieldto the tissue for electroporation of cell membranes therein. As shown inFIG. 1A, two needle-free injectors 2 and 4, each project a jet of liquid6 and 8 containing the biologically active agent. The injectors aredisposed in spaced relation to one another and in close contact with thesurface 10 of a region of tissue 12 of the subject. The portion of theinjectors in contact with the tissue surface are electrically conductiveand are in electrical connection with an electrical source (not shown),such as a pulse generator, such that electroporation is accomplished bydelivering an electrical current through the region of tissue bycompleting the circuit between the two electrically conducting injectortips. As shown in FIG. 1A, injector 2 is the active or donor electrodeand injector 4 is the counter or return electrode. In other embodiments,both injectors can act as donor electrodes. Usually, although notalways, the injectors are also in contact with the tissue surface whilethe active agent is introduced.

Another embodiment of the invention method wherein the injector isutilized to apply an electrical field to the surface of a subject isshown in FIG. 1B. In this embodiment of the invention method, at leastone injector contacts the surface of the tissue and provides anelectrical current in conjunction with one or more electrodes, such as,for example, a ring electrode(s). As shown in FIG. 1B, injector 2contacts surface 10 of a region of tissue 12 so as to act as the activeor donor electrode while charged ring electrode 14 acts as the counteror return electrode.

In yet another embodiment shown in FIG. 2, the needle-free injectorintroduces a conductive fluid as a jet through an opening 16 in an arrayelectrode 18, which contains multiple positive and negative electrodes.e.g., a micropatch electrode as described in U.S. patent applicationSer. No. 09/134,245, filed on Aug. 14, 1998, which is herebyincorporated herein in its entirety by reference). By example, theelectrode can be a meander electrode that consists of an array ofinterweaving electrode fingers with alternating polarity. The width ofindividual electrodes about 2 mm and the gap between electrodes is about0.2 mm. Alternatively, the electrode can be made of a porous materialsuch that e.g., polyacrylamide hydrogels the liquid jet from theinjector passes through the pores of the electrode to the target layersof the tissue.

Various shapes and compositions of the needle-free injector tip thatdelivers the electric pulse (or electrode, if used) can be used so longas it is capable of delivering a sufficient electric pulse as set forthherein. Optionally, at least the portion of the needle-free injectorthat is pressed against the tissue in practice of the invention methodsis insulated to protect against excess heat or burning, current leakage,shock, etc. Appropriate electric pulsing parameters are set forth hereinor can be determined using the teachings herein and, in view of theseparameters, the skilled artisan can select among various suitablematerials (e.g., ceramic, metal, etc.) and configurations (ring, soliddisc, etc.) for manufacture of the portion of the needle-free injector(and electrodes, if used) that contact the tissue to be electroporated.

In addition, to the injectors, optional electrodes, and electricalsource, the apparatus used in practice of the invention method typicallyfurther includes a means for controlling the amount of current passingfrom the device and through the contacted surface, as well as additionalcontrol elements typical of electroporation systems as are known in theart.

The liquid jets themselves from the injector(s) can be made highlyelectrically conductive by using a conductive suspension or solution ofthe agent, e.g., in an ionic solution, such as a saline solution. Asused herein, the term “conductive” means that the fluid has a specificresistivity sufficient to allow the application of an effectiveelectrical field without unacceptable heating of the liquid jetoccurring during that electrical pulse. The jets of conductive fluidthen can act, not only as liquid needles, but also as electrodes. Thus,when conductive jets of liquid are introduced into tissue, the injectordevice does not need to touch the tissue into which the active agent isintroduced. Rather, the injector can be placed in proximity to thesurface of the tissue and the conductive jet from a needle-free injectordevice in combination with such another jet, or in combination with oneor more surface electrodes is sufficient to complete the electricalcircuit through the tissue. As one of skill in the art will appreciate,therefore, in the invention methods the active agent can be introducedeither as a jet of conductive fluid from a needle-free injector (withouttouching the surface of the tissue with an electrically conductiveinjector device), or a low conductivity jet of fluid can be introducedwhile contacting the tissue surface with an electrically conductiveinjector device in any of the combinations of injector(s) andelectrode(s) described herein.

Typically in this situation, the electroporation pulse would beadministered “substantially contemporaneously” with the injection of theagent into the tissue. As used herein, the term “substantiallycontemporaneously” can mean that the electroporation pulse is deliveredduring the time that the jet remains intact (i.e., has not broken up).For example, the electroporation pulse can be timed, (e.g., mechanicallyor electronically) to coincide with the jet driving mechanism in theinjector. If electrodes are placed on the surface of the tissue for thepurpose of promoting current flow (see FIG. 1B for example), timingsensors can be incorporated into the electrodes to coordinate theelectroporation pulse and the jet driving mechanism. Alternatively, theterm “substantially contemporaneously” can mean that the needle-freeinjector is activated to inject the agent and the electric pulse isapplied to the region of skin or mucosa to be treated reasonably closetogether in time. Alternatively, an electrical current can be providedbefore or following introduction of a therapeutic agent to the tissue ofthe subject. When multiple electrical impulses are applied, the agentcan be administered in a form suitable for direct or indirectelectrotransport before or after each of the pulses, or at any timebetween the electrical pulses.

The active agent can include ionic species, molecules having chargedfunctionalities, or molecules of neutral charge. The agent may becompletely charged (i.e., 100% ionized), completely uncharged, or partlycharged and partly uncharged. Alternatively, two or more agents ofdiffering charge (or % ionization) can be combined to arrive at adesired level of charge for the combination, or an uncharged activeagent can be contained in a medium suitable for direct or indirectelectrotransport, such as a charged liquid (e.g., a solvent). Variousdegrees of ionization of the medium containing the active agent can beemployed to produce the agent in a form suitable for electrotransport.For example, the liquid medium containing the active agent can beionized from about 5% to about 95% by volume, or the liquid medium canbe ionized from about 10% to about 75%, or from about 30% to about 50%by volume.

Electroporation as utilized in the invention method is a method ofincreasing the permeability of tissue and cell membranes which allowstransport, or migration, of an agent through tissue or across cellmembranes into cells. For example, electroporation can include applyinga voltage across tissue to increase the permeability of the tissue andat least a portion of the cell membranes of cells in the tissue. If thetissue is in the presence of an agent in a form suitable forelectrotransport, as described herein, the agent migrates across thetissue and into cells of the tissue.

The electric field applied in practice of the invention method isdetermined by the nature of the tissue, the size of the selected tissue,and its location. It is desirable that the field be as homogeneous aspossible and of the correct amplitude. Excessive field strength resultsin lysing of cells, whereas a low field strength results in reducedefficacy. When the region of tissue being treated is skin or mucosa,during electroporation a voltage sufficient to cause that region of theepidermis to become electroporated is applied to the portion of theepidermis or tissue into which the active agent is introduced.

The electric pulse can be provided by any electronic device or electricpulse generator that provides an appropriate electric pulse sufficientfor introducing an active agent (e.g., a therapeutic agent) in a formsuitable for direct or indirect electrotransport into target cells. Thewaveform of the electrical signal provided by the pulse generator duringelectroporation can be an exponentially decaying pulse, a square pulse,a unipolar oscillating pulse train, or a bipolar oscillating pulsetrain, or any combination of these forms. The nominal electric fieldstrength can be from about 10 V/cm to about 20 kV/cm. The nominalelectric field strength is determined by computing the voltage betweenany two injectors (injector and one or more electrodes) divided by thedistance between the injectors (or injector and one or more electrodes).The pulse length is generally in the range from about ten μs to 100 ms.There can be any desired number of pulses, typically one to about 100pulses per second. The interval between pulse sets can be any suitabletime, such as one second. The waveform, electric field strength andpulse duration may also depend upon the type of cells or tissue and thetype of agents that are to enter the cells during electroporation.

Each pulse wave form has particular advantages; square wave form pulsesprovide increased efficiencies in transporting compounds into the cellsin comparison to exponential decay wave form pulses, and the ease ofoptimization over a broad range of voltages, as described, for example,in Saunders, Guide to Electroporation and Electrofusion, 1991, pp227–47. Preferably the waveform used is an exponential or a square wavepulse. Other wave forms such as rectangular or triangular will be knownin the art and are included herein.

The electric fields needed for in vivo cell electroporation of variouscell types are generally similar in magnitude to the fields required forcells in vitro and are well known in the art. Presently preferredmagnitudes are in the range of from 10 V/cm to about 1300 V/cm. Thehigher end of this range, over about 600 V/cm, has been verified by invivo experiments of others reported in scientific publications.

The nominal electric fields can be designated either “high” or “low.” Itis presently preferred that, when high voltage fields are used, thenominal electric field is from about 700 V/cm to 1300 V/cm and morepreferably from about 1000 V/cm to 1300 V/cm. It is presently preferredthat, when low fields are used, the nominal electric field is from about10 V/cm to 200 V/cm, and more preferably from about 25 V/cm to 75 V/cm.

In a particular embodiment of the present invention, it is presentlypreferred that when the electric field is low, the pulse length is long,i.e., the “low voltage long pulse” mode of electroporation. For example,when the nominal electric field is about 25 V/cm to 75 V/cm, it ispreferred that the pulse length is about 1 to 80 msec. For this type oflow voltage long pulse electroporation, a square wave pulse ispreferably used. Square wave electroporation systems deliver controlledelectric pulses that rise quickly to a set voltage, stay at that levelfor a set length of time (pulse length), and then quickly drop to zero.Square wave electroporation pulses have a gentler effect on the cellsthan an exponential decay pulse, and therefore, yield higher cellviability and better transformation efficiency for the electroporationof plant and mammalian tissues. Exemplary pulse generators capable ofgenerating a square pulsed electric field include, for example, theElectroSquarePorator (T820) pulse generator (BTX division ofGenetronics, Inc., San Diego, Calif.), which can generate a square waveform of up to 3000 volts and a pulse length from about 5 μsec to about99 msec. The T820 ElectroSquarePorator is active in both the HighVoltage Mode (HVM) (100–3000 Volts) and the Low Voltage Mode (LVM)(10–500 Volts). The pulse length for LVM is about 0.3 msec to 99 msecand for HVM, about 5 μsec to 99 μsec, with multiple pulsing capabilityfrom about 1 pulse to 99 pulses. Additional electroporation apparatusare commercially available and can be used in practice of the inventionmethods, for example, the ECM600 (BTX division, Genetronics, Inc.),which can generate an exponential wave form.

Although electroporation of the region of tissue treated can be priorto, simultaneously with, and/or subsequently to injection of the agent,the chemical composition of the agent will dictate the most appropriatetime to administer the agent in relation to the administration of theelectric pulse for electroporation. For example, while not wanting to bebound by a particular theory, it is believed that a drug having a lowisoelectric point (e.g., neocarcinostatin, IEP=3.78), would likely bemore effective if administered post-electroporation in order to avoidelectrostatic interaction of the highly charged drug within the field.Another group of drugs (such as bleomycin) has a very negative log P, (Pbeing the partition coefficient between octanol and water), are verylarge in size (MW about 1400), and/or are hydrophilic, therebyassociating closely with the lipid membrane. Such drugs diffuse veryslowly into a tumor cell. Therefore, in practice of the inventionmethod, drugs having such characteristics are typically administeredprior to or substantially simultaneously with the electric pulse.

In addition, certain biologically active agents may require chemicalmodification in order to facilitate more efficient entry into the cellsand/or electrotransport. For example, an agent with poor watersolubility, such as taxol, can be chemically modified using methodsknown in the art, to increase solubility in water.

The agent (and medium) may undergo electrotransport through porescreated in cell membranes (e.g., during electroporation) byelectromigration, electroosmosis, or a combination of the two.(Electroosmosis has also been referred to as electrohydrokinesis,electro-convection, and electrically-induced osmosis.) In general,electroosmosis of a therapeutic species into a tissue results from themigration of a liquid in a non-conducting capillary system in which thespecies is contained, as a result of the application of electromotiveforce to the therapeutic species reservoir., i.e., solvent flow inducedby electromigration of other ionic species (C. Morris and P. Morris,Separation Methods in Biochemistry, New York Interscience Publishers,Great Britain, 1964, pp 632, 639).

In conjunction with any of the above-described procedures, a briefperiod of iontophoresis may optionally be applied to distribute theagent between the electrodes (e.g., the injectors) before, during, orafter pulsing for electroporation. Iontophoresis is a process that canbe used to transport molecules across tissue without necessarily causingelectroporation, especially once enhanced electroporation has occurred.For iontophoresis, an electrical potential of much lower voltage andgreater duration than is used for electroporation is applied to theregion of tissue treated. For example, electroporation of the stratumcorneum is caused by large pulses (between about 50 volts and about 500volts at the electrodes), while iontophoresis is often caused byapplication of essentially steady (direct current), relatively smallvoltages (between about 0.1 Volt and about 5 Volt) or currents, whichtransport molecules through pre-existing pathways (see, for example, B.H. Sage, “Iontophoresis” in Percutaneous Penetration Enhancers E. W.Smith and H. I. Maibach, Eds., CRC Press, pp. 351–368, 1995). Therefore,in one embodiment, iontophoresis through skin tissue is practiced inconjunction with the invention methods by maintaining a constant currentof about 1 mA for 30 seconds. Those of skill in the art will know how toselect appropriate parameters to be used for iontophoresis of othertypes of tissues.

During iontophoresis, ions present in a sustained low voltage field willmigrate toward sources of opposite charge. Thus, an active agent havingat least some percent ionization will migrate towards an oppositelycharged electrode through an electroporated membrane into subcutaneous,interstitial fluids. Neutral molecules can also be moved viaiontophoresis by repeated contact of charged particles moving in onedirection, such that net transport of the neutral molecular speciesoccurs because of the transport of the electrically charged species.Iontophoresis is most efficient when the low voltage field for theiontophoresis is temporarily interrupted when the pores have retractedto a size at which the transport rate drops below a selected level (oris maintained) while a new electrical pulse having the characteristicsto induce electroporation is applied.

During iontophoresis, the skin resistance changes much more slowly, andin lesser magnitude than during electroporation, and this skinresistance behavior is believed to be due to changes of ioniccomposition of solutions within pre-existing aqueous pathways (see, forexample, S. M. Dinh, C-W. Luo and B. Berner “Upper and Lower Limits ofHuman Skin Electrical Resistance in Iontophoresis” AIChe J.39:2011–2018, 1993). Thus, the larger skin resistance duringiontophoresis means that the electric field is more confined to thesurface of the tissue than during electroporation.

The term “iontophoresis” as used herein refers to (1) the delivery ortransport of charged drugs or agents by electromigration, (2) thetransport and/or delivery of uncharged drugs or agents by the process ofelectroosmosis, (3) the transport and/or delivery of charged drugs oragents by the combined processes of electromigration and electroosmosis,and/or (4) the transport and/or delivery of a mixture of charged anduncharged drugs or agents by the combined processes of electromigrationand electroosmosis.

During the electrotransport process certain modifications or alterationsof the skin or mucosal tissue may occur, such as increased ioniccontent, hydration, dielectric breakdown, extraction of endogenoussubstances, and electroporation. Any electrically assisted transport ofspecies enhanced by modifications or alterations to a body surface(e.g., formation of pores in the skin) are also included in the termelectrotransport as used herein.

The biologically active agents and active agents introduced according tothe invention methods include drugs (e.g., chemotherapeutic agents),nucleic acids (e.g., polynucleotides), peptides and polypeptides,including antibodies and other molecules for delivery to a subject. Forexample, the polypeptide can be an antigen introduced for the purpose ofraising an immune response in the subject into whose cells it isintroduced. Alternatively, the polypeptide can be a hormone, such ascalcitonin, parathyroid hormone, erythropoietin, insulin, a cytokine, alymphokine, a growth hormone, a growth factor, and the like, or acombination of any two or more thereof. Additional illustrativepolypeptides that can be introduced into cells using the inventionmethod include blood coagulation factors and lymphokines, such as tumornecrosis factor, interleukins 1, 2 and 3, lymphotoxin, macrophageactivating factor, migration inhibition factor, colony stimulatingfactor, χ-interferon, β-interferon, χ-interferon (and subtypes thereof),and the like.

Polynucleotides or oligonucleotides that can be introduced according tothe invention methods include DNA, cDNA, and RNA sequences of all types.For example, the DNA can be double stranded DNA, single-stranded DNA,complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, andcombinations thereof. Such agents are introduced by needle-freeinjection and electroporation as described herein in an amount tomodulate cell proliferation or to elicit an immune response, eitheragainst the nucleic acid or a protein product encoded by the nucleicacid.

The polynucleotides can also be DNA constructs, such as expressionvectors, expression vectors encoding a desired gene product (e.g., agene product homologous or heterologous to the subject into which it isto be introduced), and the like. A therapeutic polypeptide (one encodinga therapeutic gene product) may be operably linked with a regulatorysequence such that the cells of the subject are transfected with thetherapeutic polypeptide, which is expressed in cells into which it isintroduced according to the invention methods. The polynucleotide mayfurther encode a selectable marker polypeptide, such as is known in theart, useful in detecting transformation of cells with active agentsaccording to the invention method.

In various embodiments of the invention method, the active agent can bea “proliferation-modulating agent,” which alters the proliferativeabilities of cells. Proliferation modulating agents include, but are notlimited to, cytotoxic agents, agents toxic or becoming toxic in thepresence of a protein, and chemotherapeutic agents. The term “cytotoxicagent” refers to a protein or other molecule having the ability toinhibit, kill, or lyse a particular cell. Cytotoxic agents includeproteins such as ricin, abrin, diphtheria toxin, saporin, or the like.In one embodiment, the cytotoxic agent is only effective when it cangain access to the cell, such as by the introduction of the agent intothe cell by needle-free injection in combination with electroporation.The introduction of such agents intracellularly, or the expression ofnucleic acids encoding polypeptides intracellularly, results ininhibition of protein synthesis or death of the cell. Illustrative toxicsubunits include the A chains of diphtheria toxin, enzymatically activeproteolytic fragments from Pseudomonas aeruginosa exotoxin-A, ricinA-chain, abrin A-chain, modeccin A-chain, and proteins having similaractivity found in various plants, such as the plants Geloniummultiflorum, Phytolacca Americana, Croton, Tiglium, Jatropha, Curcas,Momordic, Charantia, Reachan, the toxin saporin from Saponariaofficinalis (Thorpe et al. J. National Cancer Institute (1985) 75:151),the Chinese cucumber toxin, trichosanthin (Yeung et al. Intl. J. ofPeptide Protein Res. (1985) 27:325–333), and the like. Mutant species ofthe toxins also may be used, for example, CRM 45 (Boquet et al. Proc.Natl. Acad. Sci. USA (1976) 73:4449–4453).

In other embodiments, the active agent can be a “chemotherapeuticagent,” having an antitumor or cytotoxic effect. Such agents can be“exogenous” agents, which are not normally found in the subject (e.g.,chemical compounds and drugs). Chemotherapeutic agents can also be“endogenous” agents, which are native to the subject, including suitablenaturally occurring agents, such as biological response modifiers (i.e.,cytokines, hormones, and the like). Specific chemotherapeuticproliferation-modulating agents include, but are not limited todaunomycin, mitomycin C, daunorubicin, doxorubicin, 5-FU, cytosinearabinoside, colchicine, cytochalasin B, bleomycin, vincristine,vinblastine, methotrexate, and the like. Additional active agents thatact as chemotherapeutic agents are cytotoxic agents, such as thosederived from microorganism or plant sources.

Drugs contemplated for use in the invention method as the active agentinclude antibiotics such as are known in the art and chemotherapeuticagents having an antitumor or cytotoxic effect. Such drugs or agentsinclude bleomycin, neocarcinostatin, suramin, doxorubicin, carboplatin,taxol, mitomycin C, cisplatin, and the like. Other chemotherapeuticagents will be known to those of skill in the art (see for example TheMerck Index). In addition, agents that are “membrane-acting” agents canalso be introduced into cells according to the invention method.Membrane acting agents are a subset of chemotherapeutic agents that actprimarily by damaging the cell membrane, such as N-alkylmelamide,para-chloro mercury benzoate, and the like. Alternatively, thecomposition can include a deoxyribonucleotide analog, such asazidodeoxythymidine, dideoxyinosine, dideoxycytosine, gancyclovir,acyclovir, vidarabine, ribavirin, or any chemotherapeutic known to thoseof average skill in the art.

Vaccination is an effective form of preventative care against infectiousdiseases. Safe and effective vaccines are available to protect against avariety of bacterial and viral diseases. These vaccines may consist ofinactivated pathogens, recombinant or natural subunits, and liveattenuated or live recombinant microorganisms. Accordingly, in anotheraspect, an agent or composition introduced to the epidermis of a subjectcan be a vaccine, such as a vaccine that includes a polynucleotide or aprotein component.

DNA immunization, a method to induce protective immune responses using“naked” DNA, complexed DNA or encapsulated DNA, is effective as shown inU.S. Pat. No. 5,589,466. DNA immunization entails the direct, in vivoadministration of vector-based DNA or non-vector DNA that encodes theproduction of defined microbial or cellular antigens, for example, andcytokines (e.g., IL and IFN), for example. The de novo production ofthese antigens in the host's own cells results in the elicitation ofantibody and cellular immune responses that provide protection againstchallenge and persist for extended periods in the absence of furtherimmunizations. The unique advantage of this technology is its ability tomimic the effects of live attenuated vaccines without the safety andstability concerns associated with the parenteral administration of liveinfectious agents. Because of these advantages, considerable researchefforts have focused on refining in vivo delivery systems for naked DNAthat result in, for example, maximal antigen production and resultantimmune responses. Such systems also include liposomes and otherencapsulated means for delivery of DNA.

Accordingly, it is presently preferred that the DNA or RNA moleculeintroduced as a vaccine to induce a protective immune response encodesnot only the gene product (i.e., active agent) to be expressed, but alsoinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of the vaccinated subject. The vaccinepolynucleotide can optionally be included in a pharmaceuticallyacceptable carrier as described herein.

As used herein, the term “gene product” refers to a protein resultingfrom expression of a polynucleotide within the treated cell. The geneproduct can be, for example, an immunogenic protein that shares at leastan epitope with a protein from the pathogen or undesirable cell-type,such as a cancer cell or cells involved in autoimmune disease againstwhich immunization is required. Such proteins are antigens and shareepitopes with either pathogen-associated proteins, proteins associatedwith hyperproliferating cells, or proteins associated with autoimmunedisorders, depending upon the type of genetic vaccine employed. Theimmune response directed against the antigenic epitope will protect thesubject against the specific infection or disease with which theantigenic epitope is associated. For example, a polynucleotide thatencodes a pathogen-associated gene product can be used to elicit animmune response that will protect the subject from infection by thepathogen.

Likewise, a polynucleotide that encodes a gene product containing anantigenic epitope associated with a hyperproliferative disease such as,for example, a tumor-associated protein, can be used to elicit an immuneresponse directed at hyperproliferating cells. A polynucleotide thatencodes a gene product that is associated with T cell receptors orantibodies involved in autoimmune diseases can be used to elicit animmune response that will combat the autoimmune disease by eliminatingcells in which the natural form of target protein is being produced.Antigenic gene products introduced into cells as active agents accordingto the present invention may be either pathogen-associated proteins,proteins associated with hyperproliferating cells, proteins associatedwith auto-immune disorders or any other protein known to those ofaverage skill in the art.

In addition, it may be desirable to introduce into cells of a subject apolynucleotide that modulates the expression of a gene, such as anendogenous gene, in cells. The term “modulate” envisions the suppressionof expression of a gene when it is over-expressed, as well asaugmentation of expression when it is under-expressed. Where a cellproliferative disorder is associated with the expression of a gene,nucleic acid sequences that interfere with the gene's expression at thetranslational level can be used to modulate gene expression. Thisapproach introduces into the cells of a subject such active agents asantisense nucleic acid sequences, ribozymes, or triplex agents to blocktranscription or translation of a specific mRNA, either by masking thatmRNA with an antisense nucleic acid or triplex agent, or by cleaving itwith a ribozyme.

Antisense nucleic acid sequences are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acid hybridizes to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acid interferes with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely thanlarger molecules to cause problems when introduced into the target cell.The use of antisense methods to inhibit the in vitro translation ofgenes is well known in the art (Marcus-Sakura, Anal. Biochem., 172:289,1988).

Use of a short oligonucleotide sequence (i.e., “triplex agent”) to stalltranscription is known as the triplex strategy, since the oligomer windsaround double-helical DNA, forming a three-strand helix. Therefore, suchtriplex agents can be designed to recognize a unique site on a chosengene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991; Helene,C., Anticancer Drug Design, 6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences that are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences thatare 11–18 bases in length. The longer the recognition sequence, thegreater the likelihood that the sequence will occur exclusively in thetarget mRNA species. Consequently, it is preferred to employhammerhead-type ribozymes over tetrahymena-type ribozymes forinactivating a specific mRNA species, and 18-based recognition sequencesare preferable to shorter recognition sequences as active agents inpractice of the invention methods.

The active agent introduced according to the invention methods can alsobe a therapeutic peptide or polypeptide. For example, immunomodulatoryagents and other biological response modifiers can be administered forincorporation by cells. The term “biological response modifiers” ismeant to encompass substances which are involved in modifying the immuneresponse. Examples of immune response modifiers include such compoundsas lymphokines. Lymphokines include tumor necrosis factor, interleukins1, 2, and 3, lymphotoxin, macrophage activating factor, migrationinhibition factor, colony stimulating factor, and alpha-interferon,beta-interferon, and gamma-interferon, their subtypes and the like.

Also included are polynucleotides which encode metabolic enzymes andproteins, including anti-angiogenesis compounds, e.g., Factor VIII orFactor IX. The active agent introduced according to the inventionmethods can also be an antibody. The term “antibody” as used herein ismeant to include intact molecules as well as fragments thereof, such asFab and F(ab′)₂, and the like, as are known in the art.

In addition, the composition can include a detectable marker, such as aradioactive label. Alternatively, the composition can include aphotoactive modification, such as Psoralin C2. Further, the compositioncan include a phosphoramidate linkage, such as butylamidate,piperazidate, and morpholidate. Alternatively, the composition caninclude a phosphothiolate linkage or ribonucleic acid. These linkagesdecrease the susceptibility of oligonucleotides and polynucleotides todegradation in vivo.

The term “pharmaceutical agent” or “pharmaceutically active agent” asused herein encompasses any substance that will produce atherapeutically beneficial pharmacological response when administered toa subject, including both humans and animals. More than onepharmaceutically active substance may be included, if desired, in apharmaceutical composition used in the method of the present invention.

The pharmaceutically active agent can be employed in the presentinvention in various forms, such as molecular complexes orpharmaceutically acceptable salts. Representative examples of such saltsare succinate, hydrochloride, hydrobromide, sulfate, phosphate, nitrate,borate, acetate, maleate, tartrate, salicylate, metal salts (e.g.,alkali or alkaline earth), ammonium or amine salts (e.g., quaternaryammonium) and the like. Furthermore, derivatives of the activesubstances such as esters, amides, and ethers which have desirableretention and release characteristics but which are readily hydrolyzedin vivo by physiological pH or enzymes can also be employed.

As used herein, the term “therapeutically effective amount” or“effective amount” means that the amount of the biologically active orpharmaceutically active substance is of sufficient quantity and activityto induce a desired pharmacological effect. The amount of substance canvary greatly according to the effectiveness of a particular activesubstance, the age, weight, and response of the individual subject aswell as the nature and severity of the subject's condition or symptoms.Accordingly, there is no upper or lower critical limitation upon theamount of the active agent introduced into the cells of the subjectalthough it is generally a greater amount than would be delivered bypassive absorption or diffusion, but should not be so large as to causeexcessive adverse side effects to the cell or tissue containing suchcell, such as cytotoxicity, or tissue damage. The amount required fortransformation of cells will vary from cell type to cell type and fromtissue to tissue and can readily be determined by those of ordinaryskill in the art using the teachings herein. The required quantity to beemployed in practice of invention methods can readily be determined bythose skilled in the art.

In one embodiment of the invention method, the amount of active agentsuch as a nucleic acid sequence encoding a gene product introduced intothe cells is a “transforming amount.” A transforming amount is an amountof the active agent effective to modify a cell function, such as mitosisor gene expression, or to cause at least some expression of a geneproduct encoded by the nucleic acid sequence.

Introduction of active agents across the natural barrier layer of skinor mucous membrane can be enhanced by encapsulating the active agent ina controlled release vehicle or mixed with a lipid. As used herein withrespect to preparations or formulations of active agents, the term“controlled release” means that the preparation or formulation requiresat least an hour to release a major portion of the active substance intothe surrounding medium, for example, about 1–24 hours, or even longer.

Preferred controlled release vehicles that are suitable forelectrotransport are colloidal dispersion systems, which includemacromolecular complexes, nanocapsules, microcapsules, microspheres,beads, and lipid-based systems, including oil-in-water emulsions,micelles, mixed micelles, liposomes, and the like. For example, in oneembodiment, the controlled release vehicle used to contain the activeagent for injection is a biodegradable microsphere. Microspheres whereina pharmaceutically active agent is encapsulated by a coating ofcoacervates is called a “microcapsule.”

Liposomes, which may typically bear a cationic charge, are artificialmembrane vesicles useful as delivery vehicles in vitro and in vivo. Ithas been shown that large unilamellar vesicles (LUV), which range insize from about 0.2 to 4.0 μm, can encapsulate a substantial percentageof an aqueous buffer containing large macromolecules, such as DNA.

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations, making them suitable vehicles forencapsulating an active agent intended to undergo electrotransportaccording to the invention methods.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, gangliosides, and the like. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14–18carbon atoms, particularly from 16–18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoyl-phos-phatidylcholine.

Preparations suitable for electrotransport may also include a“pharmaceutically acceptable carrier.” Such carriers include sterileaqueous or non-aqueous solutions, suspensions and emulsions. Examples ofnon-aqueous solvents include propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, fixed oils,and the like. Vehicles suitable for intercellular or intracellularinjection may also include fluid and nutrient replenishers, electrolytereplenishers, such as those based on Ringer's dextrose, and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents, and inertgases, and the like.

The invention method may optionally further comprise pretreatment of thetissue surface with compounds or compositions that facilitate injectionof the active agent into cells underlying the tissue surface. Examplesof components of a composition suitable for pretreatment of theepidermis of the subject include, for example, a reducing agent, such asa charged reducing agent (e.g., DMSO) that disrupts cross linked keratinwithin keratinocytes of the epidermis. Alternatively, the epidermis canbe pretreated by application of a proteinase, such as keratinase,papain, or reducing agents or compounds, to overcome possible hindranceof DNA transport during injection and electroporation that might becaused by the dense keratin matrix of the epidermis.

As used herein, the term “subject” refers to any animal. It isenvisioned that the methods for delivering an agent into cells of asubject can be performed on any animal, including domesticated animalskept as pets, as well as animals raised as workers or as a providers orsources of food. Preferably, the subject is a human.

As used herein, the term “local,” when used in reference to an activeagent introduced by a needle-free injector according to the inventionmethod, refers to activity within the region of tissue treated (e.g.,the region electroporated). Thus, an agent injected into skin or mucosaltissue is believed to be taken up by cells underlying or contiguous withthe skin or mucosal tissue and to exert its biological or pharmaceuticalactivity within the cells of the tissue or muscle directly underlyingthe skin. Nevertheless, the skilled artisan will recognize that somebiologically active agents introduced according to the invention methodmay have a systemic effect or activity, such that, after being injectedinto a particular region of tissue according to the invention method,the agent may be distributed at least in part to other areas of thesubject, thereby producing or contributing to a systemic effect.

The invention methods for introducing an agent into cells are useful intreatment of a variety of conditions and diseases ranging from diabetesto psoriasis and baldness. Like other types of transdermal drugdelivery, the invention methods have application in treatment ofconditions that have a large potential market, such as pain management(acute and chronic), treatment of erectile dysfunction, skin aging, andthe like. For example, in one aspect, the invention method is useful intreating undesired cells. An “undesired cell” is any cell targeted forremoval due to its location, genotypic and/or phenotypic properties, andthe like. Examples of conditions exhibiting undesired cells that can betreated using the invention methods include, but are not limited to, thepresence of excess fat cells, endometrial tissue in endometriosis,excess tissue caused by psoriasis, birth marks such as port wine stains,adhesions or scar tissue from injury or surgery, moles, and the like.

The methods of the invention are useful in treating cell proliferativedisorders or other disorders of the various organ systems, particularly,for example, cells in the skin, mucosal tissue uterus, prostate andlung, and also including cells of heart, kidney, muscle, breast, colon,prostate, thymus, testis, ovary, blood vessel and the like. The term“cell proliferative disorder” refers to a disease or conditioncharacterized by inappropriate cell proliferation, and includesneoplasia. Concepts describing normal tissue growth are applicable tomalignant tissue since normal and malignant tissues can share similargrowth characteristics, both at the level of the single cell and at thelevel of the tissue. In tumors, production of new cells exceeds celldeath. For instance, a neoplastic event tends to produce an increase inthe proportion of stem cells undergoing self-renewal and a correspondingdecrease in the proportion progressing to maturation (McCulloch, E. A.,et al., Blood 59:601–608, 1982). Thus, the term “cell proliferativedisorder” denotes malignant as well as non-malignant cell populations,which often appear to differ from the surrounding tissue bothmorphologically and genotypically. Specific non-limiting examples ofnon-malignant cell proliferative disorders include warts, benignprostatic hyperplasia, skin tags, and non-malignant tumors. For example,the invention can be used to treat such cell proliferative disorders asbenign prostatic hyperplasia or unwanted genital warts by targeting theundesirable cells that characterize such conditions for removal.

The methods of the invention are advantageous in several respects.First, the invention methods allow, for example, topical treatment ofskin or mucosal lesions, such as melanoma. Such treatment is notinvasive and delivery of pharmaceutical compounds, polynucleotides orother agents can be localized to the site of the lesion. Further, theamount of agent necessary to treat a particular lesion is significantlyreduced by localized application of the agent, thereby substantiallydiminishing the cost of treatment and side effects. In addition, risk ofinfection and mechanical trauma, such as that caused by subcutaneousinjections, is avoided by using electroporation in combination withneedle-free injection. Further, risk associated with disrupting cancercells, such that they are dislodged from a primary location, therebyspreading the cancer, is lessened. In addition, systemic illnesses canbe treated by delivery of pharmaceuticals, polynucleotides, such asantisense oligonucleotides, or other agents, to control expression of atargeted gene associated with the illness over an extended period oftime.

One therapeutic application of electroporation includes needle-freeintroduction of a cytotoxic agent into tissue and electroporation of theagent into cells by applying voltage pulses between electrodes orelectrically conductive needle-free injectors disposed on opposite sidesof or within the tissue. Another therapeutic application of theinvention methods includes needle-free injection of a nucleic acidencoding a cytotoxic agent into tissue having undesirable cell types(i.e. cells proliferating in an unnatural manner) and electroporation ofthe nucleic acid into the cells of the tissue by applying voltage pulsesbetween electrodes strategically located on opposite sides or within thetissue containing undesirable cells. As disclosed herein, it ispreferred that the needle-free jet injection device itself serves as anelectrode. (See FIGS. 1A and B). However, when the injector is not usedas an electrode, caliper or surface electrodes are utilized.

The invention methods can also be used in practice of gene therapy forthe treatment of cell proliferative or immunologic disorders mediated bya particular gene or absence thereof. Such therapy would achieve itstherapeutic effect by introduction of a specific sense or antisensepolynucleotide into cells having the disorder. Polynucleotides intendedfor introduction into cells of a subject for the purpose of gene therapycan be contained in a recombinant expression vector such as a chimericvirus, or the polynucleotide can be delivered as “naked” DNA asdescribed herein.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (-MoMuLV), Harvey murinesarcoma virus (HaMuS-V), murine mammary tumor virus (-MuMTV), and RousSarcoma Virus (RSV). When the subject is a human, a vector such as thegibbon ape leukemia virus (GaLV) can be utilized. A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

The invention will now be described in greater detail by reference tothe following, non-limiting examples.

EXAMPLE 1

The following study was conducted to compare quantatively the level ofgene expression when a DNA-containing plasmid is administered byintradermal injection and by needle-free injector. Electroporation ofthe injection site at various voltage levels was used to determine andcompare electroporation enhancement of gene expression when DNA isinjected by the two methods tested.

I. Luciferase reporter gene. To quantify the level of gene expression,the luciferase reporter gene was used in the first study.

Animals. Four to six week old male and female outbred pigs weighing 20to 40 pounds were purchased from the Prairie Swine Center (University ofSaskatchewan, Saskatoon, Saskatchewan). The animals were housed andtreated in compliance with the Canadian Council for Animal Care. Animalswere randomly assigned to six groups of five animals each.

Electroporation. To determine the effect of electroporation onexpression of plasmid DNA, electroporation was performed using the BTXECM 830 Pulse Generator with the needle-free micropatch round electrodemounted on a handle (model MP 35) (Genetronics, San Diego, Calif.). Sixsquare-wave pulses were applied at 60, 70, or 80 V, with pulse durationof 60 msec, pulse interval of 200 msec, and reversal of polarity afterthree pulses.

Luciferase expression and assay. A luciferase encoding plasmid(Pmas-luc) under the control of the CMV promoter in the pMAS backbone(Krieg, A. M., et al., Proc. Natl. Acad. Sci. USA. 95:12631–6.) was agift from Dr. Heather Davis (University of Ottawa, Ontario, Canada)(Weeratna, R., et al., Antisense Nucleic Acid Drug Dev. 8:351–356).Plasmid DNA encoding luciferase was injected intradermally using a26-gauge needle or a DERMAL BIOJECT™ needle-free injection system(BioJect, Inc., Portland, Oreg.), followed by electroporation withvarious voltages. Intradermal needle injection was tested with noelectroporation and with electroporation at voltages of 60V and 80V.Needle-free using the Bioject device was tested with no electroporationand with electroporation at voltages of 60 V, 70 V, and 80 V.

For each injection, a dose of 100 μg of pMAS-luc (Weeratna et al.,supra) in 100 μl phosphate buffered saline (PBS) was administered intothe skin of the shaved abdomens (eight sites per pig), with eachtreatment group having four sites from the same pig. Theluciferase-encoding plasmid was injected at eight distinct sites, andelectroporation was applied to four of those sites. Forty-eight hoursafter the administration of the plasmid, the area surrounding eachinjection site was biopsied with an 8-mm diameter puncher at a depth ofapproximately 8-mm. Skin was homogenized in 500 μl lysis buffer(Promega, Madison, Wis.) with a POLYTRON® homogenizer (BrinkmannInstruments, Rexdale, Ontario) to produce protein extracts. Luciferaseactivity in the protein extracts was determined using Promega'sluciferase assay system. On a Packard PICOLITE® luminometer (PackardInstruments Canada LTD, Mississauga, Ontario), the bioluminescence ofeach 500-μl sample was counted for 30 seconds and recorded as relativelight units (RLUs). Untreated or PBS-injected tissues were used todetermine the background luminescence levels.

Histological examination of skin. Forty-eight hours after intradermalinjection of 100 μl PBS alone or PBS containing 100 μg of DNA using theneedle-free injection system and electroporation with 60 V, 70 V, or 80V, skin biopsies were fixed in 10% formalin. Tissues were embedded inparaffin and 2-μm sections were stained with hematoxylin/eosin (H&E).

Histological examination of tissue sections was used to determine howvoltage could influence gene expression. The results of histologicalexamination showed that increasing the voltage increases the amount oftissue damage and cellular infiltration of the skin. The optimalvoltages determined were 60V for intradermal needle injection and 70Vfor the needle-free deliveries. Thus, proper voltage selection for theDNA immunizations enhanced cellular uptake of plasmid and minimizedtissue damage.

The luciferase assay results indicated that delivery by needle-freeinjector consistently outperformed needle injections, with or withoutelectroporation. However, electroporation significantly enhanced thelevel of expression with both types of injection (FIG. 3). From theresults of these studies, it can be concluded that the optimal voltageto accompany needle injection is lower than the optimal voltage toaccompany injection with a needle-free injector.

II. GFP gene expression. To analyze localization of gene expression aplasmid encoding green fluorescent protein (GFP) was used. A plasmidencoding GFP under the control of the CMV promoter was obtained throughQuantum Biotechnologies, (Montreal, Que.). 100 μg of the plasmid in 100μl PBS was administered intradermally at the sites on the shavedabdomens of the pigs using a syringe or needle-free injector incombination with electroporation (60 V for syringe injection and 70 Vfor needle-free administration). Twenty-four hours after administrationthe injection site was biopsied using an 8-mm punch. Skin samples werefrozen in liquid nitrogen and stored at −70° C. until they weresectioned. Skin samples were cut transversally with an IEC MINITOME®microtome cryostat (Damon, Needham, Miss.) into 7-μm sections. Sectionscontaining GFP expressing cells were photographed with an OlympusAH2-RFL microscope using standard light together with blue fluorescentlight. The results were based on four skin punch biopsies per treatment.

The results of histological examination of tissue samples showed thatdelivery of plasmid by either intradermal injection or needle-freeinjector resulted in GFP expression surrounding the injection site inthe epidermis. However, delivery with the needle-free injector resultedin consistent GFP expression in skin biopsies with GFP expression in allfour biopsies; whereas the results of intradermal needle injection wasnot consistent, since GFP was only detected in one out of four biopsies.In addition, administration by needle-free injector resulted in GFPexpression in cells surrounding the hair follicles, where dendriticcells are known to be numerous. Since dendritic cells are active sitesof immunological activity, it can be expected that needle free injectionwill be a good route of administration DNA vaccines.

Electroporation influenced the localization of GFP with a much greaterdispersion of GFP in the dermis away from the injection site followingboth intradermal needle administration and needle-free injectoradministration. Delivery by needle-free injector in combination withelectroporation (70 volts) resulted in GFP expression only in the deeperlayers of the skin and was not detected under the damaged stratumcorneum. In a single isolated biopsy, GFP expression followingintradermal needle injection looked more robust and wide spread comparedto needle-free administration. All other biopsies showed thatneedle-free injection administration resulted in gene expression and,and more importantly, the gene expression was distributed in a muchwider area than resulted from intradermal needle injection.

EXAMPLE 2

To study the effects of electroporation in the context of a DNA vaccine,a DNA-prime/DNA-boost/protein-boost strategy was evaluated forenhancement of immune responses in large animals, and compared theresponses achieved following standard protein vaccination. The strategyused resembled DNA-prime/protein-boost strategies previously used innon-human primates, which yielded outstanding results for both malaria(12) and HIV vaccinations (1).

Immunization protocols. For immunization studies, animals were selectedand care for as above. Pigs were randomly assigned to six groups of fiveanimals each. The pigs were anesthetized with halothane prior to DNAinjection and electroporation and treated as follows: The animals inGroup 1 each received 250 μg pHBsAg in 100 μl 0.1 M phosphate bufferedsaline (PBS) by a dermal BIOJECT B 2000° needle-free injection device(Bioject, Inc. Portland, Oreg.) at each of two abdominal sites for atotal of 500 μg pHBsAg. The animals in Group 2 were treated identicallyto those in Group 1, except that the injection sites were also treatedwith 70 V electroporation, immediately following plasmid injection. Theanimals in Group 3 each received 250 μg pHBsAg in 100 μl PBS by anintradermal injection at each of two abdominal sites for a total of 500μg pHBsAg. The animals in Group 4 were treated identically to those inGroup 3 except that injection sites were treated with a 60 Velectroporation, immediately following plasmid administration. Animalsin Group 5 received 500 μl of the commercial hepatitis B vaccine(Engerix-B, SmithKline Beecham Pharma, Oakville, Ont.) injectedintradermally with the Bioject device in two 250 μl doses on the abdomenand Group 6 was immunized with Engerix-B by an intramucular injection.All animals were boosted with the same injection conditions after fourweeks. All treatment groups were boosted at week eight with Engerix Bvaccine by intramuscular injection for all groups except group 5, whichwas injected with Engerix using the Bioject device as previouslydescribed (Table 1).

TABLE 1 EXPERIMENTAL DESIGN 1^(st) and 2^(nd) 3^(rd) immunization³immunizations² (Engerix-B) Group¹ Vaccine Route/Method Voltage (V)Route/Method 1 pHBsAg i.d./BioJect — i.m./needle 2 pHBsAg i.d./BioJect70 V i.m./needle 3 pHBsAg i.d./needle — i.m./needle 4 pHBsAg i.d./needle60 V i.m./needle 5 Engerix-B i.d./BioJect — i.d./BioJect 6 Engerix-Bi.m./needle — i.m./needle ¹Each group consisted of five animals. ²Firstand second immunizations were administered on day zero and four weekslater. ³The third immunization was given eight weeks after the firstimmunization i.d., intradermal; i.m., intramuscular.

To assess efficacy of DNA vaccination in priming the immune system,animals in all experimental groups were boosted with a protein vaccineat week 8 post initial injection.

Measurement of humoral responses. At four, eight, and ten weeks afterthe first immunization serum was collected from anesthetized animals andcentrifuged for analysis. Anti-HBsAg antibodies were quantitativelymeasured using the AUSAB EIA Diagnostic Kit, and quantification inmilli-International Units/ml was performed in parallel with the AUSABQuantification Panel, according to the manufacturer's instructions(Abbott Laboratories, North Chicago, Ill.).

Anti-hepatitis B IgG₁ and IgG2 isotypes were identified by ELISA asfollows. IMMUNLON 2® ELISA plates (Dynex, Chantilly, Va.) were coatedwith HBsAg (BioDesign International, Saco, Me.) (1 μg/ml in 20 mMNa₂CO₃) and stored overnight at 4° C. Then, the plates were washed withphosphate-buffered saline-Tween (PBST) (PBS, 0.05% TWEEN 20®; SigmaChemical Co., St. Louis, Mo.). Serum was diluted in diluent (PBST, 0.5%gelatin) (Sigma) 20-fold, followed by serial 4-fold dilutions, andincubated overnight at 4° C. Plates were washed six times in PBST.Porcine IgG₁ and IgG₂ isotypes were detected using mouse anti-porcineantibodies specific against IgG₁ and IgG2 isotypes (Serotec, Hornby,Ontario). Following incubating at room temperature for one hour, plateswere washed six times in PBST. Anti-mouse IgG₁ biotinylated antibodies(Caltag, Toronto, Ontario), diluted in diluent, were added and incubatedfor one hour. Plates were washed six times in PB ST, andstreptavidin-alkaline phosphatase (Jackson Immuno-Research Labs, WestGrove, Pa.) was added to the plates and incubated for one hour. Thealkaline phosphatase activity was measured by the conversion ofp-nitrophenol phosphate (PNPP) (Sigma). The absorbance was read after 15to 20 minutes at 405-nm wavelength (Bio-Rad, Hercules, Calif.).

Immune Responses in Immunized Pigs. Although there was variability inthe immune response among the animals in each group described above, theanimals treated with electroporation showed better immune responses thanthe non-electroporated animals, based on both the number of animalsresponding and the level of response recorded by AUSAB (Table 2 belowand FIG. 4).

TABLE 2 Responses in Pigs to Various Hepatitis B Vaccinations¹ Number ofAnimals Responding Week 4 Week 8 Week 10 Vaccine Group AUSAB ELISA AUSABELISA AUSAB ELISA (1) DNA, b.j. 0/5 0/5 1/5 1/5 4/5 3/5 (2) DNA, b.j. +EP 0/5 0/5 2/5 5/5 5/5 5/5 (3) DNA, i.d.n. 0/5 0/5 0/5 0/5 5/5 3/5 (4)DNA, i.d.n. + EP 0/5 0/5 0/5 1/5 5/5 5/5 (5) Engerix B, b.j. 0/5 0/5 4/54/5 5/5 5/5 (6) Engerix B, i.m.n. 0/5 0/5 5/5 5/5 4/4 4/4 ¹The number ofanimals that showed an immune response out of the total number ofanimals in a group is indicated. b.j. = Bioject needle-free injection;EP = electroporation; i.d.n. = intradermal needle; i.m.n. =intramuscular needle.

The results summarized in Table 2 are consistent with the results of theluciferase experiments of Example 1 herein, which indicated thatelectroporation enhances gene expression, and that needle-free deliveryis more effective than needle delivery. These results also demonstratethe superiority of immune responses elicited with the combination ofneedle-free delivery and electroporation over responses achieved byintradermal needle injections, both with respect to the number ofanimals responding and the magnitude of the response.

As a positive control for these studies, two groups of animals wereimmunized with a subunit vaccine injected either intramuscularly withneedle and syringe (the prior art route) or intradermally with theneedle-free injector. Results of the control studies indicate that theintramuscular needle injection resulted in more robust immune responsesthan injection with the needle-free injector.

Results in Table 2 also demonstrate that even though immune responseswere undetectable in most of the animals after two rounds of DNAimmunization, the majority responded rapidly to a protein boost. Thefact that nearly all DNA immunized animals responded within two weeks ofthe protein boost demonstrates an anamnestic response, since the proteinvaccine alone did not induce immune responses 4 weeks post immunization(Table 2).

To determine whether the experimental manipulations had an impact on theantibody isotypes generated, the sera were analyzed for the presence ofanti-HBsAg IgG₁ and IgG₂ using an ELISA. As shown in FIGS. 5A–5D, inthose animals mounting an early response, most produced primarily IgG₁at 8 weeks. However, after boosting with protein, a much more balancedresponse with approximately equivalent levels of IgG₁ and IgG₂ wereevident. Additionally, the groups injected with DNA and treated withelectroporation (Groups 2 and 4) produced antibodies similar in titer tothose in the cohorts receiving multiple protein injections (Groups 5 and6). In contrast, those groups that received DNA without electroporation(Groups 1 and 3) produced antibodies of lower titer, even after theprotein boost.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the compounds and processesof this invention. Thus, it is intended that the present invention coversuch modifications and variations, provided they come within the scopeof the appended claims. Accordingly, the invention is limited only bythe following claims.

1. A method for introducing a biologically active agent into cells in aregion of tissue of a subject, said method comprising: a) using one ormore needle-free injectors of an electroporation device to inject saidagent into said region in a form suitable for electrotransport, whereinsaid electroporation device comprises: i. a needle-free injectorconfigured to serve as a first electroporation electrode when positionedin contact with tissue of the subject: ii. a second electroporationelectrode disposed in spaced relation to said first electroporationelectrode: iii. an energy source in electrical communication with saidfirst and second electroporation electrodes to energize said first andsecond electroporation electrodes to effect electroporation; and b)using said electroporation device to generate an electric fieldsufficient to electroporate cells of said region, wherein saidelectroporating is conducted prior to, simultaneously with, and/orsubsequently to injecting said agent into said region using at least oneof said needle-free injectors to introduce the biologically active agentinto said subject, whereby the combination of needle-free injection andelectroporation is sufficient to introduce the agent into the cells. 2.The method of claim 1, wherein the electric field is generated by asquare, rectangular, triangular, or exponential decay wave pulse.
 3. Themethod of claim 2, wherein the pulse is of at least 50 about V.
 4. Themethod of claim 2, wherein the pulse is from about 100 μsec to about 100msec.
 5. The method of claim 2, wherein the pulse is monopolar orbipolar.
 6. The method of claim 1, wherein the injection of the agent issimultaneous with application of the electric field wherein theneedle-free injector acts as an electrode.
 7. The method of claim 1,wherein the electroporation device comprises at least two needle-freeinjectors and the electric field is applied by contacting said regionwith at least two of the needle-free injectors in spaced apart relation,with one of the needle-free injectors serving as a donor electrode andthe other serving as a receptor electrode.
 8. The method of claim 1,wherein application of the electric field and injection of the agent issubstantially simultaneous.
 9. The method of claim 1, wherein the agentis in the form of a conductive liquid.
 10. The method of claim 9,wherein the conductive liquid is contained in a partially ionizedsolvent.
 11. The method of claim 1, wherein the agent is containedwithin a controlled release vehicle.
 12. The method of claim 1, whereinthe method is performed in vivo.
 13. The method of claim 1, wherein thesubject is a mammal.
 14. The method of claim 1, wherein the subject is ahuman.
 15. The method of claim 1, wherein the agent is a therapeuticagent.
 16. The method of claim 15, wherein the therapeutic agent isselected from the group consisting of a chemotherapeutic agent, apolynucleotide, a polypeptide, and a peptide.
 17. The method of claim15, wherein the therapeutic agent is a chemotherapeutic agent isselected from the group consisting of bleomycin, neocarcinostatin,carboplatin, cisplatin, suramin, doxorubicin, mitomycin C, cisplatin,and a combination of any two or more of the foregoing compounds.
 18. Themethod of claim 15, wherein the therapeutic agent is a nucleic acidconstruct encoding a homologous or heterologous gene product.
 19. Themethod of claim 18, wherein the cells are transfected with the nucleicacid construct so that the gene product is expressed in the cells. 20.The method of claim 18, wherein the nucleic acid construct is anexpression vector.
 21. The method of claim 20, wherein the homologous orheterologous nucleic acid of the expression vector encodes a geneproduct and is operably linked to a suitable promoter sequence.
 22. Themethod of claim 18, wherein the gene product is expressed in the cells.23. The method of claim 15, wherein the therapeutic agent is anantibody.
 24. The method of claim 15, wherein the therapeutic agent isan antibiotic.
 25. The method of claim 1, wherein the agent is selectedfrom the group consisting of a hormone, a cytokine, a lymphokine, agrowth factor, and a combination of any two or more of the foregoingcompounds.
 26. The method of claim 1, wherein the agent is injectedthrough a layer of skin into tissue underlying the skin and wherein thecells are muscle cells.
 27. The method of claim 1, wherein the agent ismixed with a lipid.
 28. The method of claim 1, wherein the tissue isskin.
 29. The method of claim 1, wherein the agent is introducedencapsulated in a liposome or mixed with a charged lipid.
 30. The methodof claim 1, wherein the agent is in a liquid and the injector forces theliquid into the tissue as a conductive or essentially non-conductiveliquid jet.
 31. The method of claim 30, wherein the liquid jet acts asan electrode.
 32. The method of claim 1, wherein the method furthercomprises applying iontophoresis to the tissue.
 33. The method accordingto claim 1, wherein the agent is a proliferation-modulating agent. 34.The method according to claim 33, wherein the proliferation-modulatingagent is selected from the group consisting of an antisense nucleic acidsequence, a ribozyme, a nucleic acid sequence, a triplex agent, and acombination of any two or more of the foregoing compounds.
 35. Themethod according to claim 33, wherein the method is intended to treat orprevent a cell-proliferative disorder or condition in the subject. 36.The method according to claim 1, wherein the active agent comprises atleast one antigenic epitope.
 37. The method according to claim 36,wherein introduction of the agent is used to generates a protectiveimmune response in the subject.
 38. A method for intramuscularadministration of a biologically active agent into cells in a region ofmuscle tissue of a subject, said method comprising: a) using one or moreneedle-free injectors of an electroporation device to introduce saidagent into said region in a form suitable for electrotransport, whereinsaid electroporation device comprises: i. at least one needle-freeinjector, wherein at one of said needle-free injectors is configured toserve as a first electroporation electrode when positioned in contactwith tissue of said subject: ii. a second electroporation electrodedisposed in spaced relation to said first electroporation electrode; andiii. an energy source in electrical communication with said first andsecond electroporation electrodes to energize said first and secondelectroporation electrodes to effect electroporation; and b) using saidelectroporation device to generate an electric field sufficient toelectroporate cells of said region, wherein said electroporating isconducted prior to, simultaneously with, and/or subsequently tointroducing said agent into said region using at least one of saidneedle-free injectors to introduce the biologically active agent intosaid-subject, whereby the combination of needle-free injection andelectroporation is sufficient to introduce the agent into the cells. 39.The method of claim 38, wherein the electric field is generated by asquare, rectangular, triangular, or exponential decay wave pulse. 40.The method of claim 39, wherein the pulse is of at least about 50 V. 41.The method of claim 39, wherein the pulse is from about 100 μsec toabout 100 msec.
 42. The method of claim 39, wherein the pulse ismonopolar or bipolar.
 43. The method of claim 38, wherein introductionof the agent is simultaneous with application of the electric field, andwherein a needle-free injector acts as an electrode.
 44. The method ofclaim 38, wherein the electric field is applied by contacting saidregion with an electroporation device that comprises at least twoneedle-free injectors in spaced apart relation, with one of theinjectors serving as a donor electrode and the other serving as areceptor electrode.
 45. The method of claim 38, wherein application ofthe electric field and injection of the agent is substantiallysimultaneous.
 46. The method of claim 38, wherein the agent is in theform of a conductive liquid.
 47. The method of claim 46, wherein theagent is contained in a partially ionized solvent.
 48. The method ofclaim 38, wherein the agent is contained within a controlled releasevehicle.
 49. The method of claim 38, wherein the method is performed invivo.
 50. The method of claim 38, wherein the subject is a mammal. 51.The method of claim 38, wherein the subject is a human.
 52. The methodof claim 38, wherein the agent is a therapeutic agent.
 53. The method ofclaim 52, wherein the therapeutic agent is selected from the groupconsisting of a chemotherapeutic agent, a polynucleotide, a polypeptideand a peptide.
 54. The method of claim 52, wherein the therapeutic agentis a chemotherapeutic agent selected from the group consisting ofbleomycin, neocarcinostatin, carboplatin, cisplatin, suramin,doxorubicin, mitomycin C, cisplatin, and a combination of any two ormore of the foregoing compounds.
 55. The method of claim 52, wherein thetherapeutic agent is a nucleic acid construct encoding a homologous orheterologous gene product.
 56. The method of claim 55, wherein the cellsare transfected with the nucleic acid construct so that the gene productis expressed in the cells.
 57. The method of claim 55, wherein thenucleic acid construct is an expression vector.
 58. The method of claim57, wherein the homologous or heterologous nucleic acid of theexpression vector encodes a gene product and is operably linked to asuitable promoter sequence.
 59. The method of claim 55, wherein the geneproduct is expressed in the cells.
 60. The method of claim 52, whereinthe therapeutic agent is an antibody.
 61. The method of claim 52,wherein the therapeutic agent is an antibiotic.
 62. The method of claim38, wherein the active agent is selected from the group consisting of ahormone, a cytokine, a lymphokine, a growth factor, and a combination ofany two or more of the foregoing compounds.
 63. The method of claim 38,wherein the agent is mixed with a lipid.
 64. The method of claim 38,wherein the agent is introduced encapsulated in a liposome or mixed witha charged lipid.
 65. The method of claim 38, wherein the agent is in aliquid and the injector forces the liquid into the tissue as aconductive or essentially non-conductive liquid jet.
 66. The method ofclaim 65, wherein the liquid jet acts as an electrode.
 67. The method ofclaim 38, wherein the method further comprises applying iontophoresis tothe tissue.
 68. The method according to claim 38, wherein the agent is aproliferation-modulating agent.
 69. The method according to claim 68,wherein the proliferation-modulating agent is selected from the groupconsisting of an antisense nucleic acid sequence, a ribozyme, a nucleicacid sequence, a triplex agent, and a combination of any two or more ofthe foregoing compounds.
 70. The method according to claim 69, whereinintroduction of the agent is performed to treat or prevent acell-proliferative disorder or condition in the subject.
 71. The methodaccording to claim 38, wherein the agent comprises at least oneantigenic epitope.
 72. The method according to claim 71, whereinintroduction of the agent is used to generates an immune response in thesubject.
 73. A method for administration of a biologically active agentinto cells in an intramucosal region of tissue of a subject, said methodcomprising: a) using one or more needle-free injectors of anelectroporation device to introduce said agent into said region in aform suitable for electrotransport, wherein said electroporation devicecomprises: i. at least one needle-free injector wherein at one of saidneedle-free injectors is configured to serve as a first electroporationelectrode when positioned in contact with tissue of said subject; ii. asecond electroporation electrode disposed in spaced relation to saidfirst electroporation electrode; and iii. an energy source in electricalcommunication with said first and second electroporation electrodes toenergize said first and second electroporation electrodes to effectelectroporation; and b) using said electroporation device to generate anelectric field sufficient to electroporate cells of said region, whereinsaid electroporating is conducted prior to, simultaneously with, and/orsubsequently to introducing said agent into said region using at leastone of said needle-free injectors to introduce the biologically activeagent into said subject, whereby the combination of needle-freeinjection and electroporation is sufficient to introduce the agent-intocells in the intramucosal region.
 74. The method of claim 73, whereinthe electric field is generated by a square, rectangular, triangular, orexponential decay wave pulse.
 75. The method of claim 74, wherein thepulse is of at least about 50V.
 76. The method of claim 74, wherein thepulse is from about 100 μsec to about 100 msec.
 77. The method of claim74, wherein the pulse is monopolar or bipolar.
 78. The method of claim73, wherein administration with the needle-free injector is simultaneouswith application of the electric field, and wherein the needle-freeinjector acts as an electrode.
 79. The method of claim 73, wherein theelectroporation device comprises at least two needle-free injectors,wherein the electric field is applied by contacting said tissues of saidregion with at least two of the needle-free injectors in spaced apartrelation, with one of the injectors serving as a donor electrode and theother serving as a receptor electrode.
 80. The method of claim 73,wherein application of the electric field and injection of the agent issubstantially simultaneous.
 81. The method of claim 73, wherein theagent is in the form of a conductive liquid.
 82. The method of claim 81,wherein the agent is contained in a partially ionized solvent.
 83. Themethod of claim 73, wherein the agent is contained within a controlledrelease vehicle.
 84. The method of claim 73, wherein the method isperformed in vivo.
 85. The method of claim 73, wherein the subject is amammal.
 86. The method of claim 73, wherein the subject is a human. 87.The method of claim 73, wherein the agent is a therapeutic agent. 88.The method of claim 87, wherein the therapeutic agent is selected fromthe group consisting of a chemotherapeutic agent, a polynucleotide, apolypeptide, and a peptide.
 89. The method of claim 87, wherein thetherapeutic agent is a chemotherapeutic agent selected from the groupconsisting of bleomycin, neocarcinostatin, carboplatin, cisplatin,suramin, doxorubicin, mitomycin C, cisplatin, and a combination of anytwo or more of the foregoing compounds.
 90. The method of claim 87,wherein the therapeutic agent is a nucleic acid construct encoding ahomologous or heterologous gene product.
 91. The method of claim 90,wherein the cells are transfected with the nucleic acid construct sothat the gene product is expressed in the cells.
 92. The method of claim90, wherein the nucleic acid construct is an expression vector.
 93. Themethod of claim 92, wherein the homologous or heterologous nucleic acidof the expression vector encodes a gene product and is operably linkedto a suitable promoter sequence.
 94. The method of claim 93, wherein thegene product is expressed in the cells.
 95. The method of claim 87,wherein the therapeutic agent is an antibody.
 96. The method of claim87, wherein the therapeutic agent is an antibiotic.
 97. The method ofclaim 87, wherein the agent is selected from the group consisting of ahormone, a cytokine, a lymphokine, a growth factor, and a combination ofany two or more of the foregoing compounds.
 98. The method of claim 87,wherein the agent is mixed with a lipid.
 99. The method of claim 87,wherein the agent is introduced encapsulated in a liposome or mixed witha charged lipid.
 100. The method of claim 87, wherein the agent is in aliquid and the injector forces the liquid into the tissue as aconductive or essentially non-conductive liquid jet.
 101. The method ofclaim 100, wherein the liquid jet acts as an electrode.
 102. The methodof claim 87, wherein the method further comprises applying iontophoresisto the tissue.
 103. The method according to claim 87, wherein the agentis a proliferation-modulating agent.
 104. The method according to claim87, wherein the proliferation-modulating agent is selected from thegroup consisting of an antisense nucleic acid sequence, a ribozyme, anucleic acid sequence, a triplex agent, and a combination of any two ormore of the foregoing compounds.
 105. The method according to claim 87,wherein introduction of the active agent is in treatment or preventionof a cell-proliferative disorder or condition in a subject in needthereof.
 106. The method according to claim 87, wherein the agentcomprises at least one antigenic epitope.
 107. The method according toclaim 87, wherein introduction of the agent is used to generates animmune response in the subject.